Primary gene transcripts of eukaryotes contain introns, which are removed during processing by splicing machinery. Biochemical studies In vitro have identified a specific pathway in which introns are recognised and spliced out. This occurs by progressive formation of spliceosomal complexes designated as E, A, B, and C. The composition and structure of these spliceosomal conformations have been characterised in many detail. In contrast, transitions between the complexes and the intermediates of these reactions are currently less clear. We have previously isolated a novel 35S U5 snRNP from HeLa nuclear extracts. The protein composition of this particle differed from the canonical 20S U5 snRNPs but was remarkably similar to the activated B* spliceosomes. Based on this observation we have proposed a hypothesis that 35S U5 snRNPs represent a dissociation product of the spliceosome after both transesterification reactions are completed. Here we provide experimental evidence that 35S U5 snRNPs are generated from the activated B* spliceosomes during In vitro splicing.